Measuring hormone levels

Most hormones are present in the circulating blood in extremely low concentrations, some as low as one millionth of a microgram (one picogram) per mL. Due to the extremely minute levels, measuring hormone concentrations proved difficult in early endocrinology. In the mid 20th century, however, a revolutionary technique called radioimmunoassay was developed, making possible the measurement of hormones and their end products.

Radioimmunoassay is a procedure that occurs in four steps. In the first step, an antibody specific for a hormone is developed. In the second step, this antibody is mixed with fluid from two types of samples: 1. a sample of fluid from the organism to be tested and 2. several samples of purified standard hormone which has been marked with a radioactive isotope. It is important to note that the amount of the antibody used has to be less than the amount needed to bind the combined quantity of hormone in the fluid. This antibody deficiency creates competition among antibody binding sites, and is the basis upon which the relative concentration of each sample can be determined. Following equilibrium, the third step involves analyzing the relative binding using radioactive counting techniques. Large amount of radioactive hormone binding is indicative of relatively small hormone concentrations in the organism’s fluid and vice versa. The fourth step involves compiling several experimental results and plotting the “standard curve” relationship between radioactive concentration and organism hormone concentrations.

When compared with test assays, “standard curves” of hormone concentrations can elucidate the unknown concentrations within an error of $10$10-$15$15 percent, allowing the assay of billionths or even trillionths of a gram of hormone.